Sensitive detection and separation of fluorescent derivatives using capillary electrophoresis with laser-induced fluorescence detection with 532Â nm Nd:YAG laser

In this work, a home-built CELIF instrument with diode pumped frequency-doubled continuous wave Nd:YAG excitation laser with feedback power regulation (532 nm) was constructed. The suitability of this type of laser for LIF detection in a separation method was found excellent. A limit of detection (LOD) (S/N=3) of 2×10−13 mol/l was achieved with rhodamine B, which is comparable to those obtained using similar instruments with Ar+ laser [Y.F. Cheng, N.J. Dovichi, Science 242 (1988) 562, E.S. Yeung et al., J. Chromatogr. 608 (1992) 73]. LOD of a protein derivatized according to modified procedures [M.J. Little et al., Anal. Chim. Acta 339 (1997) 279, A. Chersi et al., Biochim. Biophys. Acta 1336 (1997) 83] was determined. Detection of the derivatives was found to be limited by insufficient reaction recovery at low analyte concentration, chemical noise, separation efficiency and quality of the derivatizing reagent rather than by the detector performance. As a consequence, a huge gap between the detection ability of CELIF instruments and LOD determined in real samples is revealed.